Download Protein synthesis in the Liver and the Urea Cycle

Protein synthesis in the
Liver and the Urea Cycle
Dr NC Bird
This lecture will consider the features
of how nitrogen is removed from
amino acids and converted to urea
and the major proteins synthesised
by the liver.
In this overview we can see how the
amino acid pool is added to from
dietary protein and how the
intracellular proteins circlulate these
free amino acids in a continuous
cycle of synthesis and breakdown.
Excess amino acids are metabolised
(not stored for use as potential
energy because this can be done
more efficiently using other energy
sources). The carbon skeleton, as
an α-keto acid, is fed into the citric
acid cycle to be incorporated into
glucose production whilst the
ammonia is largely excreted,
although some is used in the
biosynthesis of amine containing
substances.
In the liver; input of amine groups
comes from dietary amino acids,
alanine from muscle and glutamine
from muscle. The α-ketoglutarate /
glutamine pair are the most common
of the donor /acceptor pairs . Serum
levels of the aminotransferase
enzymes are used clinically as
indicators of liver cell damage (these
reactions occur in the hepatocytes) –
the most clinically useful being
Aspartate aminotransferase (AST)
and alanine aminotransferase (ALT)
–higher levels indicating that more
enzyme has leaked from the
damaged hepatocyte.
(a)
α ketoglutarate acting as an amine
acceptor giving us L-glutamate and
an α keto acid. PLP represents
pyridoxal phosphate, which is the
metabolically active form of Vitamin
B6 and is a co-factor in all of these
reactions.
(b)
In the bottom reaction alanine is
deaminated to pyruvate - part of the
glucose alanine cycle.
Glutamate Dehydrogenase reaction
This reaction goes both ways using
NAD in the forward reaction and
NADP the other way. The forward
reaction generates α ketoglutarate
which is fed into the citric acid cycle
and so hepatocytes are capable of
upregulating GDH activity at times of
energy depletion (at a cellular level
at least). So as the illustration
shows, ADP / GDP drive the reaction
forward – because they represent
the ‘low energy’ i.e. ATP or GTP
have been ‘used’ by the cell so in
order to reconstitute them substrate
for the citric acid cycle is generated
and ATP is replenished. When ATP
concentration is high then glutamate
is formed and because it is an amino
acid, it is available for incorporation
into protein.
Glutamine Synthetase reaction
A reaction which produces an amino
acid – glutamine - suitable for
incorporation into proteins. Its
principal role appears to be as a
circulating ammonia carrier,
consequently it is the most abundant
amino acid found in blood. In this
state it is a neutral, non-toxic
compound which passes readily
through cell membranes.
Glutaminase
In most land animals glutamine is
carried in the blood to the liver. As is
the case for glutamate, the amino
nitrogen is released only within the
mitochondria by this enzyme
glutaminase.
Glutamine synthetase is a cytosolic
enzyme whereas glutaminase is a
mitochondrial enzyme, so they are
located in separate compartments
which ensures that the liver is neither
a net consumer or producer of
glutamine. The differences in
cellular location of these two
enzymes allows the liver to
scavenge ammonia that hasn’t been
incorporated into urea and so
ammonia concentration is controlled
by either incorporation into urea or
glutamine.
Glucose alanine cycle
In muscle, alanine is the principal
ammonia scavenger and transporter.
Glutamate collects the ammonia,
the enzyme alanine
aminotransferase (ALT)
transaminates the amino group from
glutamate, forming α ketoglutarate,
and the amino group gets attached
to pyruvate, formed from glycolysis,
making alanine. This gets
transported in the blood, taken up by
the liver where the reverse reaction
occurs and the ammonia gets
converted to urea. Pyruvate is recycled into glucose.
This is a superb illustration of
economy of effort in solving two
problems with one cycle. Moving
carbon atoms of pyruvate, as well as
excess ammonia, from muscle to
liver as alanine. Then in the liver,
alanine yielding pyruvate – the
starting block for gluconeogenesis,
and releasing ammonia for
conversion into urea. The energetic
burden of gluconeogenesis being
imposed on the liver rather than
muscle, so that muscle ATP can be
devoted to muscle contraction.
The urea cycle.
Discovered by Hans Krebs and Kurt
Henseleit in this university in 1932.
Henseleit was a medical student
here also.
The essential features of the urea
cycle reactions and their metabolic
regulation are as follows:Arginine either from the diet or
protein breakdown, is cleaved by
arginase generating urea and
ornithine. In subsequent reactions a
new urea is built on the ornithine
(from ammonia and CO2 ) making
citrulline. This, in turn, is
reconfigured into arginine. The
enzymes responsible for this are
found partly in the mitochondria and
partly in the cytosol (like
glutaminase/glutamine synthase).
The reactions of one turn of the cycle
consume 3 ATP equivalents and a
total of 4 high energy nucleotide
PO4= . Urea is the only compound
generated by the cycle: all other
components are re-cycled. The
energy consumed by urea
production is generated in the
production of the cycle
intermediates.
Control of the cycle is via up or down
regulation of the enzymes
responsible for urea formation. So
with long term changes in the
quantity of dietary protein,
upregulation in the order of 20 times
has been demonstrated. This can
be due to either increased intake as
with body builders – high protein low
fat diets - or in starvation because
muscle proteins are being broken
down with the amino acid carbon
skeletons providing the energy.
Thus the amount of ammonia that
must be excreted increases.
Defects in the urea cycle.
Absence of any of the enzymes
involved in urea synthesis is not
compatible with life. Deficiencies in
any one of them can occur and are
well described in the textbooks, but
in terms of their likely impact on your
future clinical practice they are
insignificant and will probably be
dealt with in your paediatrics
modules. The common thread to
them all is the elevation of ammonia
levels in the blood.
Neurotoxicity associated with
Ammonia.
Elevated blood ammonia is seen in
severe liver disease, whether it be as
a result of liver failure due to
infection, toxicity or substantial
surgical resection. This is something
that is seen in the clinical practice
(not uncommonly) and since
ammonia is neurotoxic, is one of
those things that staff are conscious
of when a patient with liver disease
becomes confused or comatose .
The mechanism for the increase in
ammonia is basically the same in all
cases. In simple terms, the blood
doesn’t get exposed to enough liver
parenchymal cells to have the
ammonia removed. This can be due
either to the fact that there simply
aren’t enough living, metabolising
cells because they’ve been killed off
by the disease process be it viral or
toxic. Or because, in cirrhosis for
example, the resistance to blood flow
through the liver is so great (because
of fibrosis) that the blood by-passes
the liver by flowing through large
collateral veins. It therefore gets
delivered to the brain directly.
Ammonia crosses the blood-brain
barrier readily. Once inside it is
converted to glutamate via glutamate
dehydrogenase and so depletes the
brain of α ketoglutarate. As
ketoglutarate falls, so does
oxaloacetate and ultimately citric
acid cycle activity stops, leading to
irreparable cell damage and neural
cell death.
Albumin leaves the circulation via the
interstitium to the lymph system and
back to the circulation via the
thoracic duct. Between 4-5% of total
intravascular albumin extravasates
per hour. This rate of movement is
known as the Transcapillary Escape
rate and is determined by :1.Capillary and interstitial free
albumin concentration.
2.Capillary permeability to albumin
3.Movement of solute/solvent
4.Electrical charges across the
capillary wall (albumin has a strongly
negative charge)
The biological half-life in the
circulation is around 16-18 hours
Functions
1.Binding and transport
***************************************
Major proteins produced by the liver
which have significant extra-hepatic
roles.
Albumin
A highly soluble, single polypeptide
protein with a MW of 66000. Around
9-12 g produced per day with the
rate of production being controlled by
changes in colloid osmotic pressure
and osmolality of the extravascular
liver space. Production can be
increased by 2 to 3 fold when
necessary.
There are 4 binding sites on albumin
which have varying specificity for
different substances. Competitive
binding of drugs may occur at either
the same site or different sites
(causing conformational changes
which affect other binding sites).
The drugs that are important for
albumin binding are; warfarin,
NSAIDS, midazolam, thiopentone.
2.Maintenance of colloid osmotic
pressure
Colloid osmotic pressure is the term
used to describe the effective
osmotic pressure across blood
vessel walls which are permeable to
electrolytes but not larger molecules.
It is almost entirely due to plasma
proteins.
The Starling equation
Net Driving Pressure = Kf x [(Pc – Pi)
– rc(pc – pi)]
Kf is the filtration coefficient
Pc – hydrostatic pressure in the
capillary
Pi – hydrostatic pressure in the
interstitium
rc – the reflection coefficient is a
correction factor used to correct the
magnitude of the measured gradient
to take account of the ‘effective
oncotic pressure’ (ie in systems
where protein concs are low e.g.CSF
the rc will be close to 1 whereas in
the liver lymph the conc of protein is
high and the value is close to 0
pc – oncotic pressure in the capillary
pi- oncotic pressure in the
interstitium.
So net fluid flux is proportional to
this driving pressure. Also, the
capillary hydrostatic pressure falls
along the capillary from the arteriolar
to the venous end and so the driving
pressure decreases.
3.
Free Radicals
Albumin has a large number of
sulphydryrl groups. These thiols are
able to scavenge free radicals –
nitrogen and oxygen species. This
may be particularly important in
sepsis.
4.
Anticoagulant effects.
Albumin has both anticoagulant and
antithrombotic effects, both of which
are poorly understood. They may be
related to its binding of nitric oxide
radicals which would have the effect
of inhibiting inactivation and
therefore prolonging the biological
half – lives.
What causes albumin to decrease?
1.Decreased synthesis.
In liver disease or in large
resections, the functional mass of
the liver is reduced and therefore its
ability to synthesise proteins is
likewise reduced.
2.Increased catabolism
Very slow decline in levels because
it is synthesised at such a rate and
synthesis can be upregulated
several fold.
3.Increased loss
* Nephrotic syndrome – where there
is increased glomerular permeability
which allows proteins to filter through
and so loss of up to several grams of
protein per day can occur
* Exudative loss in burns. Extensive
tissue damage with concomittent
damage to the capillaries and
therefore loss of protein through the
wall.
* Haemorrhage
* Gut loss
A rare syndrome – protein losing
enteropathy in which the wall of the
gut is unusually permeable to large
molecules. More common however
is ulcerative colitis where the site of
ulceration is the site of increased
permeability
Consequences of decreased serum
albumin
1.Decreased colloid oncotic pressure
Decreased colloid oncotic pressure
and oedema formation (oedema
being the accumulation of fluid in the
interstitial space). The formation of
oedema is determined by the rate of
fluid flux and the clearance of fluid by
the lymphatics.
Pre-eclampsia – the association of
hypertension, proteinuria and
oedema in pregnancy- there is a
paradoxical decrease in plasma
volume and capillary leak syndrome.
Burns / Trauma
Loss through leaky capillaries
although burns patients can also
develop a protein losing enteropathy
Sepsis
In critical illness there is increased
leakage of albumin and decreased
synthesis. There is a stronger
correlation between colloid oncotic
pressure and total protein because
the decreased albumin synthesis is
compensated for by increased
synthesis of acute phase proteins.
2.Decreased ligand binding
Drug kinetics are altered, similarly
hormone transport can be affected.
Disease processes associated with
low serum albumin
Malnutrition – a diet with a high
proportion of low-grade cereal
proteins (such as maize) can lead to
a deficiency in the essential amino
acid lysine which in turn leads to a
decrease in protein synthesis. In
contrast starvation does not lead to
low albumin levels – protein from
muscle is used as the energy
source.
Liver disease
Renal diseases – albumin loss
through the glomerulus and to a
small extent during dialysis.
Increased capillary permeability –
possibly due to bacterial endotoxins
and cytotoxic T cells. There is also a
profound reduction in plasma
albumin associated with marked fluid
shifts.
There is controversy about the use
of albumin in clinical practice.
Previously studies have shown a
correlation between low serum
albumin and mortality. Therefore the
obvious thing to do, it would seem,
would be to raise serum albumin
levels by albumin infusion. However,
these ‘normalisation ‘ regimes have
not proved to be effective – in fact
one meta- analysis has suggested a
higher mortality rate in critically ill
patients treated with albumin. It is
still used by some – others adopt the
‘’treat the reason for the capillary
leakage and the patient will get
better’ strategy.
Clotting proteins produced by the
liver
With the exception of von Willebrand
Factor (VIII) the liver is responsible
for the production of all of the
coagulation proteins. Many are
serine proteases which require
activation
Clotting cascade
In this example Factor 9a (a serine protease
itself) activates Factor 10 by cleavage of the
heavy chain.
Many have a γ carboxyglutamic acid
residue which requires Vitamin K
(also stored in the liver) for
conversion.